Hot-Melt Extrusion: from Theory to Application in Pharmaceutical Formulation-Where Are We Now?

Hot-melt extrusion (HME) is a globally recognized, robust, effective technology that enhances the bioavailability of poorly soluble active pharmaceutical ingredients and offers an efficient continuous manufacturing process. The twin-screw extruder (TSE) offers an extremely resourceful customizable mixer that is used for continuous compounding and granulation by using different combinations of conveying elements, kneading elements (forward and reverse configuration), and distributive mixing elements. TSE is thus efficiently utilized for dry, wet, or melt granulation not only to manufacture dosage forms such as tablets, capsules, or granule-filled sachets, but also for designing novel formulations such as dry powder inhalers, drying units for granules, nanoextrusion, 3D printing, complexation, and amorphous solid dispersions. Over the past decades, combined academic and pharmaceutical industry collaborations have driven novel innovations for HME technology, which has resulted in a substantial increase in published articles and patents. This article summarizes the challenges and models for executing HME scale-up. Additionally, it covers the benefits of continuous manufacturing, process analytical technology (PAT) considerations, and regulatory requirements. In summary, this well-designed review builds upon our earlier publication, probing deeper into the potential of twin-screw extruders (TSE) for various new applications.

Intranasal delivery of thin-film freeze-dried monoclonal antibodies using a powder nasal spray system.

Monoclonal antibodies (mAbs) administered intranasally as dry powders can be potentially applied for the treatment or pre-exposure prevention of viral infections in the upper respiratory tract. However, a method to transform the mAbs from liquid to dry powders suitable for intranasal administration and a device that can spray the dry powders to the desired region of the nasal cavity are needed to fully realize the potentials of the mAbs. Herein, we report that thin-film freeze-dried mAb powders can be sprayed into the posterior nasal cavity using Aptar Pharma's Unidose (UDS) Powder Nasal Spray System. AUG-3387, a human-derived mAb that neutralizes the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was used in the present study. First, we prepared thin-film freeze-dried AUG-3387 powders (i.e., TFF AUG-3387 powders) from liquid formulations containing different levels of mAbs. The TFF AUG-3387 powder with the highest solid content (i.e., TFF AUG-3387C) was then chosen for further characterization, including the evaluation of the plume geometry, spray pattern, and particle size distribution after the powder was sprayed using the UDS Powder Nasal Spray. Finally, the deposition patterns of the TFF AUG-3387C powder sprayed using the UDS Powder delivery system were studied using 3D-printed nasal replica casts based on the CT scans of an adult and a child. It is concluded that it is feasible to intranasally deliver mAbs as dry powders by transforming the mAbs into dry powders using thin-film freeze-drying and then spraying the powder using a powder nasal spray system.

Characterization of mRNA Lipid Nanoparticles by Electron Density Mapping Reconstruction: X-ray Scattering with Density from Solution Scattering (DENSS) Algorithm.

PURPOSE: This study aimed to test the feasibility of using Small Angle X-ray Scattering (SAXS) coupled with Density from Solution Scattering (DENSS) algorithm to characterize the internal architecture of messenger RNA-containing lipid nanoparticles (mRNA-LNPs). METHODS: The DENSS algorithm was employed to construct a three-dimensional model of average individual mRNA-LNP. The reconstructed models were cross validated with cryogenic transmission electron microscopy (cryo-TEM), and dynamic light scattering (DLS) to assess size, morphology, and internal structure. RESULTS: Cryo-TEM and DLS complemented SAXS, revealed a core-shell mRNA-LNP structure with electron-rich mRNA-rich region at the core, surrounded by lipids. The reconstructed model, utilizing the DENSS algorithm, effectively distinguishes mRNA and lipids via electron density mapping. Notably, DENSS accurately models the morphology of the mRNA-LNPs as an ellipsoidal shape with a "bleb" architecture or a two-compartment structure with contrasting electron densities, corresponding to mRNA-filled and empty lipid compartments, respectively. Finally, subtle changes in the LNP structure after three freeze-thaw cycles were detected by SAXS, demonstrating an increase in radius of gyration (Rg) associated with mRNA leakage. CONCLUSION: Analyzing SAXS profiles based on DENSS algorithm to yield a reconstructed electron density based three-dimensional model can be a useful physicochemical characterization method in the toolbox to study mRNA-LNPs and facilitate their development.

Inhaled JAK Inhibitor GDC-0214 Nanoaggregate Powder Exhibits Improved Pharmacokinetic Profile in Rats Compared to the Micronized Form: Benefits of Thin Film Freezing.

Asthma is a common chronic disease affecting the airways in the lungs. The receptors of allergic cytokines, including interleukin (IL)-4, IL-5, and IL-13, trigger the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway, which involves the pathogenesis of asthma. GDC-0214 is a JAK inhibitor that was developed as a potent and selective target for the treatment of asthma, specifically targeting the lungs. While inhaled GDC-0214 is a promising novel treatment option against asthma, improvement is still needed to achieve increased potency of the powder formulation and a reduced number of capsules containing powder to be inhaled. In this study, high-potency amorphous powder formulations containing GDC-0214 nanoaggregates for dry powder inhalation were developed using particle engineering technology, thin film freezing (TFF). A high dose per capsule was successfully achieved by enhancing the solubility of GDC-0214 and powder conditioning. Lactose and/or leucine as excipients exhibited optimum stability and aerosolization of GDC-0214 nanoaggregates, and aerosolization of the dose was independent of air flow through the device between 2 and 6 kPa pressure drops. In the rat PK study, formulation F20, which contains 80% GDC-0214 and 20% lactose, resulted in the highest AUC0-24h in the lungs with the lowest AUC0-24h in the plasma that corresponds to a 4.8-fold higher ratio of the lung-to-plasma exposures compared to micronized crystalline GDC-0214 powder administered by dry powder inhalation. Therefore, GDC-0214 nanoaggregates produced by TFF provided an improved dry powder for inhalation that can lead to enhanced therapeutic efficacy with a lower risk of systemic toxicity.

3D printing of biologics-what has been accomplished to date?

Three-dimensional (3D) printing is a promising approach for the stabilization and delivery of non-living biologics. This versatile tool builds complex structures and customized resolutions, and has significant potential in various industries, especially pharmaceutics and biopharmaceutics. Biologics have become increasingly prevalent in the field of medicine due to their diverse applications and benefits. Stability is the main attribute that must be achieved during the development of biologic formulations. 3D printing could help to stabilize biologics by entrapment, support binding, or crosslinking. Furthermore, gene fragments could be transited into cells during co-printing, when the pores on the membrane are enlarged. This review provides: (i) an introduction to 3D printing technologies and biologics, covering genetic elements, therapeutic proteins, antibodies, and bacteriophages; (ii) an overview of the applications of 3D printing of biologics, including regenerative medicine, gene therapy, and personalized treatments; (iii) information on how 3D printing could help to stabilize and deliver biologics; and (iv) discussion on regulations, challenges, and future directions, including microneedle vaccines, novel 3D printing technologies and artificial-intelligence-facilitated research and product development. Overall, the 3D printing of biologics holds great promise for enhancing human health by providing extended longevity and enhanced quality of life, making it an exciting area in the rapidly evolving field of biomedicine.

Viability of Lactobacillus acidophilus in Thin-Film Freeze-Dried Powders Filled in Delayed-Release Vegetarian Capsules in a Simulated Gastric Fluid.

Previously, we have shown that thin-film freeze-drying can be applied to prepare dry powders of bacteria such as Lactobacillus acidophilus. Herein, we tested the viability of L. acidophilus in thin-film freeze-dried powders (TFF powders) filled in delayed-release vegetarian capsules in a simulated gastric fluid (SGF) consisting of 0.1N hydrochloric acid and sodium chloride. Initially, we determined the water removal rate from frozen thin films on relatively larger scales (i.e., 10-750 g). We then prepared and characterized two TFF powders of L. acidophilus with either sucrose and maltodextrin or sucrose and hydroxypropyl methylcellulose acetate succinate (HPMC-AS), a pH-sensitive polymer, as excipients and evaluated the viability of the bacteria after the TFF powders were filled in delayed-release vegetarian capsules and the capsules were incubated in the SGF for 30 min. On 10-750 g scales and at the settings specified, water removal from frozen thin films was faster than from slow shelf-frozen bulk solids. When the L. acidophilus in sucrose and HPMC-AS TFF powder was filled into a delayed-release capsule that was placed into another delayed-release capsule, the bacterial viability reduction after incubation in the SGF can be minimized to within 1 log in colony forming unit (CFU). However, for the L. acidophilus in sucrose and maltodextrin TFF powder, even in the capsule-in-capsule dosage form, bacterial CFU reduction was > 2 logs. TFF powders of live microorganisms containing an acid-resistant material in capsule-in-capsule delayed-release vegetarian capsules have the potential for oral delivery of those microorganisms.

Can the Oral Bioavailability of the Discontinued Prostate Cancer Drug Galeterone Be Improved by Processing Method? KinetiSol® Outperforms Spray Drying in a Head-to-head Comparison.

Galeterone, a novel prostate cancer candidate treatment, was discontinued after a Phase III clinical trial due to lack of efficacy. Galeterone is weakly basic and exhibits low solubility in biorelevant media (i.e., ~ 2 µg/mL in fasted simulated intestinal fluid). It was formulated as a 50-50 (w/w) galeterone-hypromellose acetate succinate spray-dried dispersion to increase its bioavailability. Despite this increase, the bioavailability of this formulation may have been insufficient and contributed to its clinical failure. We hypothesized that reformulating galeterone as an amorphous solid dispersion by KinetiSol® compounding could increase its bioavailability. In this study, we examined the effects of composition and manufacturing technology (Kinetisol and spray drying) on the performance of galeterone amorphous solid dispersions. KinetiSol compounding was utilized to create galeterone amorphous solid dispersions containing the complexing agent hydroxypropyl-ß-cyclodextrin or hypromellose acetate succinate with lower drug loads that both achieved a ~ 6 × increase in dissolution performance versus the 50-50 spray-dried dispersion. When compared to a spray-dried dispersion with an equivalent drug load, the KinetiSol amorphous solid dispersions formulations exhibited ~ 2 × exposure in an in vivo rat study. Acid-base surface energy analysis showed that the equivalent composition of the KinetiSol amorphous solid dispersion formulation better protected the weakly basic galeterone from premature dissolution in acidic media and thereby reduced precipitation, inhibited recrystallization, and extended the extent of supersaturation during transit into neutral intestinal media.

Feasibility of intranasal delivery of thin-film freeze-dried, mucoadhesive vaccine powders.

Intranasal vaccination by directly applying a vaccine dry powder is appealing. However, a method that can be used to transform a vaccine from a liquid to a dry powder and a device that can be used to administer the powder to the desired region(s) of the nasal cavity are critical for successful intranasal vaccination. In the present study, using a model vaccine that contains liposomal monophosphoryl lipid A and QS-21 adjuvant (AdjLMQ) and ovalbumin (OVA) as a model antigen, it was shown that thin-film freeze-drying can be applied to convert the liquid vaccine containing sucrose at a sucrose to lipid ratio of 15:1 (w/w) into dry powders, in the presence or absence of carboxymethyl cellulose sodium salt (CMC) as a mucoadhesive agent. Ultimately, the thin-film freeze-dried AdjLMQ/OVA vaccine powder containing 1.9% (w/w) of CMC (i.e., TFF AdjLMQ/OVA/CMC1.9% powder) was selected for additional evaluation because the TFF AdjLMQ/OVA/CMC1.9% powder was mucoadhesive and maintained the integrity of the antigen and the physical properties of the vaccine. Compared to the TFF AdjLMQ/OVA powder that did not contain CMC, the TFF AdjLMQ/OVA/CMC1.9% powder had a lower moisture content and a higher glass transition temperature. In addition, the TFF AdjLMQ/OVA/CMC1.9% thin films were relatively thicker than the TFF AdjLMQ/OVA thin films without CMC. When sprayed with Aptar Pharma's Unidose Powder Nasal Spray System (UDSP), the TFF AdjLMQ/OVA powder and the TFF AdjLMQ/OVA/CMC1.9% powder generated similar particle size distribution curves, spray patterns, and plume geometries. Importantly, after the TFF AdjLMQ/OVA/CMC1.9% powder was sprayed with the UDSP nasal device, the integrity of the OVA antigen and the AdjLMQ liposomes did not change. Finally, a Taguchi L4 orthogonal array was applied to identify the optimal parameters for using the UDSP device to deliver the TFF AdjLMQ/OVA/CMC1.9% powder to the middle and lower turbinate and the nasopharynx regions in both adult and child nasal replica casts. Results from this study showed that it is feasible to apply the thin-film freeze-drying technology to transform a nasal vaccine candidate from liquid to a dry powder and then use the UDSP nasal device to deliver the vaccine powder to the desired regions in the nasal cavity for intranasal vaccination.

Pre-Processing a Polymer Blend into a Polymer Alloy by KinetiSol Enables Increased Ivacaftor Amorphous Solid Dispersion Drug Loading and Dissolution.

This study compares the effects of pre-processing multiple polymers together to form a single-phase polymer alloy prior to amorphous solid dispersion formulation. KinetiSol compounding was used to pre-process a 1:1 (w/w) ratio of hypromellose acetate succinate and povidone to form a single-phase polymer alloy with unique properties. Ivacaftor amorphous solid dispersions comprising either a polymer, an unprocessed polymer blend, or the polymer alloy were processed by KinetiSol and examined for amorphicity, dissolution performance, physical stability, and molecular interactions. A polymer alloy ivacaftor solid dispersion with a drug loading of 50% w/w was feasible versus 40% for the other compositions. Dissolution in fasted simulated intestinal fluid revealed that the 40% ivacaftor polymer alloy solid dispersion reached a concentration of 595 µg/mL after 6 h, 33% greater than the equivalent polymer blend dispersion. Fourier transform infrared spectroscopy and solid-state nuclear magnetic resonance revealed changes in the ability of the povidone contained in the polymer alloy to hydrogen bond with the ivacaftor phenolic moiety, explaining the differences in the dissolution performance. This work demonstrates that the creation of polymer alloys from polymer blends is a promising technique that provides the ability to tailor properties of a polymer alloy to maximize the drug loading, dissolution performance, and stability of an ASD.

Predicting Glass-Forming Ability of Pharmaceutical Compounds by Using Machine Learning Technologies.

Low aqueous solubility is a common and serious challenge for most drug substances not only in development but also in the market, and it may cause low absorption and bioavailability as a result. Amorphization is an intermolecular modification strategy to address the issue by breaking the crystal lattice and enhancing the energy state. However, due to the physicochemical properties of the amorphous state, drugs are thermodynamically unstable and tend to recrystallize over time. Glass-forming ability (GFA) is an experimental method to evaluate the forming and stability of glass formed by crystallization tendency. Machine learning (ML) is an emerging technique widely applied in pharmaceutical sciences. In this study, we successfully developed multiple ML models (i.e., random forest (RF), XGBoost, and support vector machine (SVM)) to predict GFA from 171 drug molecules. Two different molecular representation methods (i.e., 2D descriptor and Extended-connectivity fingerprints (ECFP)) were implemented to process the drug molecules. Among all ML algorithms, 2D-RF performed best with the highest accuracy, AUC, and F1 of 0.857, 0.850, and 0.828, respectively, in the testing set. In addition, we conducted a feature importance analysis, and the results mostly agreed with the literature, which demonstrated the interpretability of the model. Most importantly, our study showed great potential for developing amorphous drugs by in silico screening of stable glass formers.

The applications of machine learning to predict the forming of chemically stable amorphous solid dispersions prepared by hot-melt extrusion.

Amorphous solid dispersion (ASD) is one of the most important strategies to improve the solubility and dissolution rate of poorly water-soluble drugs. As a widely used technique to prepare ASDs, hot-melt extrusion (HME) provides various benefits, including a solvent-free process, continuous manufacturing, and efficient mixing compared to solvent-based methods, such as spray drying. Energy input, consisting of thermal and specific mechanical energy, should be carefully controlled during the HME process to prevent chemical degradation and residual crystallinity. However, a conventional ASD development process uses a trial-and-error approach, which is laborious and time-consuming. In this study, we have successfully built multiple machine learning (ML) models to predict the amorphization of crystalline drug formulations and the chemical stability of subsequent ASDs prepared by the HME process. We utilized 760 formulations containing 49 active pharmaceutical ingredients (APIs) and multiple types of excipients. By evaluating the built ML models, we found that ECFP-LightGBM was the best model to predict amorphization with an accuracy of 92.8%. Furthermore, ECFP-XGBoost was the best in estimating chemical stability with an accuracy of 96.0%. In addition, the feature importance analyses based on SHapley Additive exPlanations (SHAP) and information gain (IG) revealed that several processing parameters and material attributes (i.e., drug loading, polymer ratio, drug's Extended-connectivity fingerprints (ECFP) fingerprints, and polymer's properties) are critical for achieving accurate predictions for the selected models. Moreover, important API's substructures related to amorphization and chemical stability were determined, and the results are largely consistent with the literature. In conclusion, we established the ML models to predict formation of chemically stable ASDs and identify the critical attributes during HME processing. Importantly, the developed ML methodology has the potential to facilitate the product development of ASDs manufactured by HME with a much reduced human workload.

Aerosolizable Plasmid DNA Dry Powders Engineered by Thin-film Freezing.

PURPOSE: This study was designed to test the feasibility of using thin-film freezing (TFF) to prepare aerosolizable dry powders of plasmid DNA (pDNA) for pulmonary delivery. METHODS: Dry powders of pDNA formulated with mannitol/leucine (70/30, w/w) with various drug loadings, solid contents, and solvents were prepared using TFF, their aerosol properties (i.e., mass median aerodynamic diameter (MMAD) and fine particle fraction (FPF)) were determined, and selected powders were used for further characterization. RESULTS: Of the nine dry powders prepared, their MMAD values were about 1-2 µm, with FPF values (delivered) of 40-80%. The aerosol properties of the powders were inversely correlated with the pDNA loading and the solid content in the pDNA solution before TFF. Powders prepared with Tris-EDTA buffer or cosolvents (i.e., 1,4-dioxane or tert-butanol in water), instead of water, showed slightly reduced aerosol properties. Ultimately, powders prepared with pDNA loading at 5% (w/w), 0.25% of solid content, with or without Tris-EDTA were selected for further characterization due to their overall good aerosol performance. The pDNA powders exhibited a porous matrix structure, with a moisture content of < 2% (w/w). Agarose gel electrophoresis confirmed the chemical integrity of the pDNA after it was subjected to TFF and after the TFF powder was actuated. A cell transfection study confirmed that the activity of the pDNA did not change after it was subjected to TFF. CONCLUSION: It is feasible to use TFF to produce aerosolizable pDNA dry powder for pulmonary delivery, while preserving the integrity and activity of the pDNA.

Accelerated water removal from frozen thin films containing bacteria.

Freeze-drying, or lyophilization, is widely used to produce pharmaceutical solids that contain temperature-sensitive materials. Herein, using Escherichia coli as a model live organism, whose viability in dry powders is highly sensitive to the water content in the powders, we demonstrated that the drying rate from the frozen thin films generated by thin-film freezing (TFF) is significantly faster than from the bulk frozen solids in conventional shelf freeze-drying. This is likely because the loosely stacked frozen thin films provided a larger solid-air interface and the low thickness of the thin films provided a low mass transfer resistance. The highly porous microstructure and high specific surface area of the thin-film freeze-dried powders may also be related to the faster drying observed. Moreover, we demonstrated that TFF can be applied to produce dry powders of E. coli, a Gram-negative bacterium, or Lactobacillus acidophilus, a Gram-positive bacterium, with minimum bacterial viability loss (i.e., within one log reduction). It is concluded that the TFF technology is promising in accelerating water removal from frozen samples.

Oral Delivery of Niclosamide as an Amorphous Solid Dispersion That Generates Amorphous Nanoparticles during Dissolution.

Niclosamide is an FDA-approved anthelmintic that is being studied in clinical trials as a chemotherapeutic and broad-spectrum antiviral. Additionally, several other applications are currently in the preclinical stage. Unfortunately, niclosamide is a poorly water soluble molecule, with reduced oral bioavailability, which hinders its use for new indications. Moreover, niclosamide is a poor glass former; in other words, the molecule has a high tendency to recrystallize, and it is virtually impossible to generate a stable amorphous solid employing the neat molecule. Previously, our group reported the development of an amorphous solid dispersion (ASD) of niclosamide (niclosamide ASD) that generates nanoparticles during its dissolution, not only increasing niclosamide's apparent solubility from 6.6 ± 0.4 to 481.7 ± 22.2 µg/mL in fasted state simulated intestinal fluid (FaSSIF) but also its oral bioavailability 2.6-fold in Sprague-Dawley rats after being administered as a suspension. Nevertheless, niclosamide ASD undergoes recrystallization in acidic media, and an enteric oral dosage form is needed for its translation into the clinic. In this work, we further characterized the nanoparticles that generated during the dissolution of the niclosamide ASD. Cryogenic transmission electron microscopy (Cryo-TEM) and wide-angle X-ray scattering (WAXS) revealed that the nanoparticles were amorphous and had a particle size of ~150 nm. The oral dosage forms of niclosamide ASD were formulated using commercial enteric capsules (Capsuline® and EudracapTM) and as enteric-coated tablets. The enteric dosage forms were tested using pH-shift dissolution and acid-uptake tests, using the USP type II dissolution apparatus and the disintegration apparatus, respectively. The capsules exhibited a higher percentage of weight gain, and visual rupture of the Capsuline capsules was observed. Eudracap capsules protected the formulation from the acidic media, but polymer gelling and the formation of a nondispersible plug were noted during dissolution testing. In contrast, enteric-coated tablets protected the formulation from acid ingress and maintained the performance of niclosamide ASD granules during dissolution in FaSSIF media. These enteric-coated tablets were administered to beagle dogs at a niclosamide dose of 75 mg/kg, resulting in plasma concentrations of niclosamide higher than those reported in the literature using solubilized niclosamide at a higher dose (i.e., 100 mg/kg). In summary, an enteric oral dosage form of niclosamide ASD was formulated without hindering the generation of nanoparticles while maintaining the increase in the niclosamide's apparent solubility. The enteric-coated tablets successfully increased the niclosamide plasma levels in dogs when compared to a niclosamide solution prepared using organic solvents.

Pharmaceutical dry powders of small molecules prepared by thin-film freezing and their applications - A focus on the physical and aerosol properties of the powders.

Thin-film freeze-drying is a dry powder engineering technology that involves in a rapid thin-film freezing (TFF) process followed by lyophilization to remove solvent. TFF process has been successfully used to produce pharmaceutical powders of the small and large molecule drugs as well as live organisms. This paper provides a comprehensive review of the pharmaceutical applications of TFF powders of small molecule drugs intended for pulmonary delivery or oral administration. The powders produced by the TFF process often exhibited unique physical properties such as amorphous morphology, high porosity, brittle matrix structure, and high specific surface area, and the powders also often contain loosely connected micron or submicron particles or aggregates. The TFF process parameters and formulation compositions directly affect the physical properties of the powders. These physical properties render TFF powders desirable aerodynamic properties for efficient pulmonary delivery by oral inhalation and help enhance the dissolution of poorly water-soluble small molecule drugs in the powders and thus improve their bioavailability after oral administration.

Emerging Artificial Intelligence (AI) Technologies Used in the Development of Solid Dosage Forms.

Artificial Intelligence (AI)-based formulation development is a promising approach for facilitating the drug product development process. AI is a versatile tool that contains multiple algorithms that can be applied in various circumstances. Solid dosage forms, represented by tablets, capsules, powder, granules, etc., are among the most widely used administration methods. During the product development process, multiple factors including critical material attributes (CMAs) and processing parameters can affect product properties, such as dissolution rates, physical and chemical stabilities, particle size distribution, and the aerosol performance of the dry powder. However, the conventional trial-and-error approach for product development is inefficient, laborious, and time-consuming. AI has been recently recognized as an emerging and cutting-edge tool for pharmaceutical formulation development which has gained much attention. This review provides the following insights: (1) a general introduction of AI in the pharmaceutical sciences and principal guidance from the regulatory agencies, (2) approaches to generating a database for solid dosage formulations, (3) insight on data preparation and processing, (4) a brief introduction to and comparisons of AI algorithms, and (5) information on applications and case studies of AI as applied to solid dosage forms. In addition, the powerful technique known as deep learning-based image analytics will be discussed along with its pharmaceutical applications. By applying emerging AI technology, scientists and researchers can better understand and predict the properties of drug formulations to facilitate more efficient drug product development processes.

Entrapment of air microbubbles by ice crystals during freezing exacerbates freeze-induced denaturation of proteins.

Freezing techniques are an essential part of biologics manufacturing processes, yet the formation of ice/water interfaces can impart detrimental effects on proteins. However, the absence of chemical and structural differences between ice and liquid water poses the question as to why ice can destabilize proteins. We hypothesize that the destabilizing stress of the ice-liquid water interface does not originate from the ice-water system itself but rather from the air microbubbles present during the freezing process. As the temperature decreases, the dissolved air is expelled from the ice crystal lattices in the form of microbubbles and is subsequently trapped by the advancing ice front. This newly formed air-water interface represents an additional interfacial area for the proteins to be adsorbed onto and denatured. The result showed that freezing at âˆ¼ 1 K/s led to the formation of small circular microbubbles with diameters ranging from 100 µm to 500 µm. In contrast, slower freezing resulted in the formation of larger, elongated millimeter-size bubbles. The reduction of the number of microbubbles was carried out by the deaeration process using agitation under reduced pressure at 20 kPa. The resulting deaerated (i.e., low dissolved air) protein samples were frozen and monitored for the formation of subvisible aggregates using micro-flow imaging (MFI). The results demonstrated that deaerating the samples prior to intermediate freezing (i.e., TFF) reduced the number of aggregates for both highly surface-active and low surface-active proteins (lactoferrin and bovine IgG, respectively). This reduction was more pronounced in spray freeze drying (SFD) than thin-film freezing (TFF), and less apparent in conventional lyophilization.

Aggregation of Lactoferrin Caused by Droplet Atomization Process via a Two-Fluid Nozzle: The Detrimental Effect of Air-Water Interfaces.

Biological macromolecules, especially therapeutic proteins, are delicate and highly sensitive to denaturation from stresses encountered during the manufacture of dosage forms. Thin-film freeze-drying (TFFD) and spray freeze-drying (SFD) are two processes used to convert liquid forms of protein into dry powders. In the production of inhalable dry powders that contain proteins, these potential stressors fall into three categories based on their occurrence during the primary steps of the process: (1) droplet formation (e.g., the mechanism of droplet formation, including spray atomization), (2) freezing, and (3) frozen water removal (e.g., sublimation). This study compares the droplet formation mechanism used in TFFD and SFD by investigating the effects of spraying on the stability of proteins, using lactoferrin as a model. This study considers various perspectives on the denaturation (e.g., conformation) of lactoferrin after subjecting the protein solution to the atomization process using a pneumatic two-fluid nozzle (employed in SFD) or a low-shear drop application through the nozzle. The surface activity of lactoferrin was examined to explore the interfacial adsorption tendency, diffusion, and denaturation process. Subsequently, this study also investigates the secondary and tertiary structure of lactoferrin and the quantification of monomers, oligomers, and, ultimately, aggregates. The spraying process affected the tertiary structure more negatively than the tightly woven secondary structure, resulting in the peak position corresponding to the tryptophan (Trp) residues red-shifting by 1.5 nm. This conformational change can either (a) be reversed at low concentrations via relaxation or (b) proceed to form irreversible aggregates at higher concentrations. Interestingly, when the sample was allowed to progress into micrometer-sized aggregates, such a dramatic change was not detected using methods such as size-exclusion chromatography, polyacrylamide gel electrophoresis, and dynamic light scattering at 173°. A more complete understanding of the heterogeneous protein sample was achieved only through a combination of 173 and 13° backward and forward scattering, a combination of derived count rate measurements, and microflow imaging (MFI). After studying the impact of droplet formation mechanisms on aggregation tendency of lactoferrin, we further investigated two additional model proteins with different surface activity: bovine IgG (serving as a non surface-active negative reference), and ß-galactosidase (another surface-active protein). The results corroborated the lactoferrin findings that spray-atomization-related stress-induced protein aggregation was much more pronounced for proteins that are surface active (lactoferrin and ß-galactosidase), but it was minimal for non-surface-active protein (bovine IgG). Finally, compared to the low-shear dripping used in the TFFD process, lactoferrin underwent a relatively fast conformational change upon exposure to the high air-water interface of the two-fluid atomization nozzle used in the SFD process as compared to the low shear dripping used in the TFFD process. The interfacial-induced denaturation that occurred during spraying was governed primarily by the size of the atomized droplets, regardless of the duration of exposure to air. The percentage of denatured protein population and associated activity loss, in the case of ß-galactosidase, was determined to range from 2 to 10% depending on the air-flow rate of the spraying process.